Inhibition of Meiotic Progression in Mouse Oocytes by CCB02

Linlin Li

Wuhan University

Chun So *

National Institute of Biological Sciences

Jul 2025 - Aug 2025

Abstract

CCB02 binds the CPAP-binding site on tubulin, disrupting centrosome function in somatic cells. Mouse oocyte meiosis, which is centrosome-independent, was treated with increasing CCB02 concentrations (0–10 mM) for 7 hours. Spindle assembly was impaired at all doses, and higher concentrations delayed meiotic progression, increasing the proportion of cells at earlier stages. These results demonstrate a dose-dependent inhibitory effect of CCB02 on oocyte meiosis, providing insights into spindle assembly regulation during oogenesis.

Introduction

CCB02 was identified and optimized to selectively bind to the CPAP-binding site on tubulin, thereby competitively inhibiting the interaction between CPAP and tubulin, allowing CPAP to recruit greater amounts of its other interacting proteins, such as pericentriolar material (PCM), to interphase centrosomes. This disrupts normal centrosome clustering, activates the spindle assembly checkpoint (SAC), and ultimately prevents cell division.

However, mouse oocyte meiosis relies on a centrosome-independent mechanism of spindle assembly, in which CPAP does not localize to the spindle. To better understand this process, we investigated the effect of CCB02 during mouse oocyte meiosis. Our results showed that mouse oocyte meiosis was inhibited by CCB02 in a dose-dependent manner, providing new insights into the regulation of spindle assembly during oogenesis.

Methods

GV-stage mouse oocytes were isolated and treated with gradient concentrations of CCB02 (0 mM, 2.5 mM, 5 mM, and 10 mM) for 7 hours, followed by fixation, immunofluorescence staining, and confocal microscopy imaging.

Isolation and quality inspection of GV-stage mouse oocytes.
Isolation and quality inspection of GV-stage mouse oocytes.

Results

Oocytes treated with a low concentration of CCB02 (2.5 mM) for 7 hours progressed to GVBD stage, whereas those treated with a high concentration (10 mM) remained at the GV stage; in both conditions, spindle assembly was defective.

In vehicle-treated oocytes, components of acentriolar microtubule-organizing centers (aMTOCs) γ-tubulin and PCNT were properly concentrated at spindle poles, and spindle assembly was normal. In contrast, CCB02-treated oocytes showed disrupted γ-tubulin and PCNT localization, accompanied by defective spindle assembly.
In vehicle-treated oocytes, components of acentriolar microtubule-organizing centers (aMTOCs) γ-tubulin and PCNT were properly concentrated at spindle poles, and spindle assembly was normal. In contrast, CCB02-treated oocytes showed disrupted γ-tubulin and PCNT localization, accompanied by defective spindle assembly.

Increasing CCB02 concentration slowed oocyte development, raising the proportion of cells at earlier meiotic stages. Our findings complement previous work by demonstrating the effects of CCB02 in oocytes that undergo centrosome-independent spindle assembly, highlighting its relevance to meiotic progression during oogenesis.

Oocytes were predominantly at the M1 stage after vehicle treatment (92.9%), at the GVBD stage after 2.5 mM CCB02 treatment (92.9%), and at the GV stage after 10 mM CCB02 treatment (81.3%).
Oocytes were predominantly at the M1 stage after vehicle treatment (92.9%), at the GVBD stage after 2.5 mM CCB02 treatment (92.9%), and at the GV stage after 10 mM CCB02 treatment (81.3%).